The large and growing problem of drug resistance of Mtb in humans requires new approaches to determine the mechanism of multi-drug resistance and markers of the resistant state in humans. Using 116 drug resistant Mtb isolates, we used whole genome sequencing to identify Mtb mutations stastically associated to drug resistance during natural infection of humans. This unbiased and broad approach identified all of the known genes that promote drug resistance and identified 11 genes of known function with no known connection to drug resistance. Three of these genes (ppsA, polyketide synthase 12 and polyketide synthase 3) produce polykefides with inter-related functions in outer membrane integrity of Mtb. An independent, mass spectromety analysis of molecules changed in a strain evolving drug resistance in vivo identified mannosyl phosphomycoketide, the product of polyeketide synthase 12, as being inversely correlated with resistance. Based on independent genetic and metabolomic discovery of altered polyketide synthesis function in drug resistant strains, we will test a new model in which altered polykefide function controls cell wall integrity and drug resistance. Using patient derived strains from Lima, Peru, we seek independent validation of altered polykefide biosynthetic genes in human populations. In the laboratory, we will test whether polykefide synthase genes and the lipids they produce influence molecular remodeling of the cell wall, Mtb permeability changes and drug resistance. To determine if genes showing strong statistical associations are actually causal of the drug resistant state or altered fitness, we will test measure the cell wall components and an in vivo fitness of polyketide deficient and drug-resistanct Mtb. Finally, taking advantage of whole organism screens that identify the particular bacterial metabolites changed in polyketide-deficient or drug resistant states, we will identify the changed molecules to give insight to the mechanism of resistance and to identify biomarkers of the drug resistant state.